The long term goals of this project are to elucidate a novel mechanism by which microtubule (MT) invasions of dendritic spines provide a direct route for the transport of postsynaptic material into spines via MT-based molecular motor proteins. In doing so we will identify the specific motor proteins and their cargos which are involved in this process. Additionally, we will investigate the role kinesin motors and MTs play in fragile X syndrome (FXS), a disease that affects spine morphology and composition, resulting in intellectual and developmental disabilities. We have previously demonstrated that MT invasions into spines increase the amount of postsynaptic density-95 (PSD- 95) protein and that spines enlarge when invaded by MTs in an NMDA receptor-dependent manner. However, to date, there is no direct mechanism which demonstrates the precise role MTs play in synaptic plasticity. We hypothesize that the molecular motor protein kinesin can directly move cargos into spines during MT invasions and is essential for the transport of postsynaptic material critical for the formation and plasticity of mature functional synapses. In Aim 1, we will test the hypothesis that kinesin motors directly enter spines via MT invasions and determine which kinesin family members are involved in this process. We will use multi-wavelength live-cell total internal fluorescence microscopy (TIRFM) to visualize fluorescently tagged kinesin-1, 2 and 3 family members and fluorescently labeled tubulin and/or EB3 in mature rat hippocampal neurons. In Aim 2 we will test the hypothesis that specific kinesin family members associate with and transport unique cargos into dendritic spines in an activity based manner. We will test this hypothesis using a variety of techniques to visualize both fluorescently-labeled kinesin family members and cargos, and determine which kinesin family members are responsible for transporting specific cargos into spines prior to and after bouts of synaptic activity (e.g. by BDNF-induced plasticity or activation of NMDA receptors). In Aim 3 we will test the hypothesis that neurons derived from human induced pluripotent stem cells (hiPSC) from FXS patients have defects in MT and kinesin invasions into dendritic spines. Together these studies will provide fundamental insights into a novel mechanism underlying synaptic plasticity and may have implications for a devastating developmental disease.